Catheter-associated urinary tract infection (CAUTI) is one of the leading causes of nosocomial infections and the outcomes range from patient discomfort, pyelonephritis, morbidity and, in rare cases, death. Numerous pathogens cause CAUTI by forming biofilms on inserted catheters including Gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, Gram-positive cocci, and fungi. The rise of P. aeruginosa induced CAUTI is of particularly concern because P. aeruginosa biofilms are highly refractory to oral antibiotic therapy. The molecular mechanisms that allow bacteria to form biofilms on the catheter during CAUTI are not well understood. To uncover those molecular events, we have utilized P. aeruginosa as a model pathogen in which in vitro biofilm formation is well characterized. We have recently developed a murine model of CAUTI. Using this murine CAUTI model, we have published results showing that isogenic polysaccharide-deficient mutants colonized catheters as efficiently as parental PA14 and PA01 strains. These surprising results indicate that P. aeruginosa formed during CAUTI is fundamentally different from biofilms characterized in laboratory conditions. Our recent RNA-seq data reveal that urine induces the down-regulation of the quorum signaling (QS) pathway and the up-regulation of the Entner- Doudoroff metabolic pathway. These results led us to hypothesize that changes to P. aeruginosa gene expression in response to host urine contributes to CAUTI. We will address our hypothesis by completing the following aims: 1) Identify the mechanism and requirement of urine suppression of QS during CAUTI, 2) Determine the requirement for Entner-Doudoroff pathway during CAUTI, and 3) Determine the selective pressure of chronic CAUTI on P. aeruginosa. Results from our proposed studies will characterize the molecular mechanism of QS repression by urine and determine the contribution of differentially regulated genes to CAUTI. Together, findings from these experiments will advance our understanding of chronic, biofilm-based infections by bacterial pathogens.